Factor VIII polypeptide having factor VIII:C activity

ABSTRACT

Factor VIII polypeptides having FVIII:C activity that contain modifications in the A3 and/or C1 and/or C2 domains of the sequence of the light chain of Factor VIII, characterized by the binding affinity to low density lipoprotein receptor protein, and methods for producing the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/986,281 filed Nov. 12, 2004, now U.S. Pat. No. 7,544,660, which is acontinuation of U.S. application Ser. No. 09/831,679 filed May 10, 2001,now U.S. Pat. No. 6,919,311, which is a continuation of PCT/AT99/00272filed Nov. 10, 1999, the entireties of which are hereby incorporated byreference.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing in computer readable format is included herewith.

FIELD OF INVENTION

The present invention relates to modified Factor VIII (FVIII)polypeptides having FVIII:C activity and containing modifications in theA3 and/or C1 and/or C2 domains of the sequence of the light chain ofFactor VIII. Furthermore, the present invention relates to nucleic acidmolecules encoding such modified Factor VIII polypeptides, vectors andhost cells containing said nucleic acid molecules, and compositionscontaining said Factor VIII polypeptide for use in the treatment ofhemorrhagenic disturbances.

BACKGROUND OF THE INVENTION

Hemostasis involves the interaction of various hemostatic reactionroutes finally leading to thrombus formation. Thrombi are deposits ofblood components on the surface of the vascular wall that mainly consistof aggregated blood platelets and insoluble cross-linked fibrin. Fibrinformation is the result of the restricted proteolysis of fibrinogen bythrombin, a coagulation enzyme. Thrombin is the end product of thecoagulation cascade, a succession of zymogen activations occurring onthe surfaces of activated blood platelets and leucocytes, and a varietyof vascular cells (for a survey, cf. K. G. Mann et al., Blood, 1990,Vol. 76, pp. 1-16).

A key function in the coagulation cascade resides in the activation ofFactor X by the complex of activated Factor IX (Factor IXa) andactivated Factor VIII (Factor VIIIa). A deficiency or a dysfunction ofthe components of this complex is associated with the blood diseaseknown as hemophilia (J. E. Sadler & E. W. Davie: Hemophilia A,Hemophilia B, and von Willebrand's Disease, in G. Stamatoyannopoulos etal., (Eds.): The molecular basis of blood diseases. W.B. Saunders Co.,Philadelphia, 1987, pp. 576-602). Hemophilia A is related to adeficiency of Factor VIII activity, whereas Hemophilia B is related to aFactor IX deficiency. Current treatment consists of a replacementtherapy using pharmaceutical preparations comprised of the normalcoagulation factor. Of these thrombopathies, Hemophilia A occurs morefrequently, affecting approximately one out of 10,000 men. Replacementtherapy in Hemophilia A patients involves the repeated administration ofpreparations containing normal Factor VIII by intravenous infusion. Theinterval between the infusions is a function of the degradation of theFactor VIII activity in blood circulation. The half-life of the FactorVIII activity after an infusion differs from one individual to another,ranging from 10 to 30 hours. Thus, a prophylactic therapy requires aninfusion every two to three days. This constitutes a heavy load on thelife of hemophilic patients, in particular, if the venous access hasbecome difficult due to local citratization following frequent needlepunctures for intravenous infusions.

It would be particularly advantageous if the frequency of infusionscould be lowered by using Factor VIII having extended half-lives. Thehalf-life of Factor VIII may be extended by interfering with themechanism of Factor VIII degradation (clearance), for instance, byreducing the affinity of Factor VIII to receptors that are essential toits clearance, either directly by modifying Factor VIII on its bindingsite(s) for the clearance receptors concerned, or indirectly by usingcompounds interfering with the interaction of Factor VIII with thosereceptors. However, the design of such agents has so far been impeded bynot knowing the Factor VIII clearance mechanism, the cell receptorsinvolved in this process, and the molecular sites involved in the FactorVIII receptor interaction.

There is limited knowledge in the molecular field as to the clearancemechanism of Factor VIII. The Factor VIII protein is synthesized as asingle chain polypeptide comprising 2332 amino acids and having thetypical domain structure A1-A2-B-A3-C1-C2 (G. A. Vehar et al., Nature,Vol. 312, 1984, pp. 337-342; J J. Toole et al., Nature, Vol., 312, 1984,342-347). Factor VIII enters the blood circulation as a heterodimericcomplex of heavy and light chains as a result of intracellularendoproteolytic processing. The light chain comprises the amino acidresidues 1649-2332 and contains the A3-C1-C2 domains. The heavy chaincontains the domains A1-A2-B (residues 1-1648) and is heterogenic due tothe limited proteolysis in a number of positions within the B domain.The Factor VIII heterodimer has no biological activity, but theheterodimer becomes active as a cofactor of the enzyme Factor IXa afterproteolytic activation by thrombin or Factor Xa. Proteolysis affectsboth the heavy chain and the light chain of Factor VIII (M. J. S. H.Donath et al., J. Biol. Chem., Vol. 270, 1995, pp. 3648-3655), leadingto the cleavage of an amino-terminal fragment from the light chain and abreak of domain connection sites within the heavy chain (between domainsA1-A2 and A2-B). The activated cofactor, Factor VIIIa, is a heterotrimercomprised of the A1 domain, the A2 domain and the light chain includingdomains A3-C1-C2.

It is well known in the art that the half-life of the non-activatedFactor VIII heterodimer strongly depends on the presence of vonWillebrand Factor, which exhibits a strong affinity to Factor VIII (yetnot to Factor VIIIa) and serves as a carrier protein (J. E. Sadler andE. W. Davie: Hemophilia A, Hemophilia B and von Willebrand's disease, inG. Stamatoynnopoulos et al. (Eds.): The molecular basis of blooddiseases. W.B. Saunders Co., Philadelphia, 1987, pp. 576-602). It isknown that patients suffering from von Willebrand's disease type 3, whodo not have a detectable von Willebrand Factor in their bloodcirculation, also suffer from a secondary Factor VIII deficiency. Inaddition, the half-life of intravenously administered Factor VIII inthose patients is 2 to 4 hours, which is considerably shorter than the10 to 30 hours observed in Hemophilia A patients.

From these findings results that Factor VIII tends to a rapid clearancefrom the blood circulation and that this process is to some extentinhibited by complexation with its natural carrier, von WillebrandFactor. Nevertheless, its half-life remains undesirably short.

Recently, it has been indicated in a preliminary report that Factor VIIIactivated by thrombin binds to low density lipoprotein receptor protein(“LRP”) (A. Yakhyaev et al., Blood, Vol. 90 (Suppl. 1), 1997, 126-I(Abstract). This abstract describes the cell absorption and thedegradation of Factor VIII fragments activated by thrombin and reportsthat the A2 domain, unlike the two other subunits of the Factor VIIIaheterotrimer, interacts with cell-bound LRP. The authors have suggestedthat binding of the A2 domain to LRP further destabilizes the looseinteraction of the A2 domain in the Factor VIIIa heterotrimer andthereby downwardly regulating Factor VIIIa activity.

It is known that LRP is one of the receptors that are involved in theclearance of various proteins. LRP in this field is also known as thealpha2-macroglobulin receptor, belonging to the family of low densitylipoprotein (LDL) receptors. It is comprised of two non-covalentlyconnected polypeptide chains: an alpha chain (515 kd) and a β-chain (85kd) [for a review refer to D. K. Strickland et al., FASEB J Vol. 9,1995, pp. 890-898]. LRP is. a multi-ligand receptor for lipoprotein andproteinase catabolism. The β-chain includes a transmembrane domain and ashort cytoplasmatic tail which is essential to endocytosis. The alphachain functions as a large ectodomain and includes three types ofrepeats: epidermal growth factor-like domains, Tyr-Trp-Thr-Asp sequencesand LDL receptor class A domains. These class A domains are present infour separate clusters, clusters I (2 domains), II (8 domains), III (20domains) and IV (11 domains). It has been shown that these clusters areinvolved in ligand binding. LRP is expressed in a plurality of tissuessuch as the placenta, lungs, brain, and liver. In the liver, LRP ispresent on parenchyma cells and Kupffer cells. Moreover, LRP isexpressed in a plurality of cell types such as fibroblasts, smoothmuscle cells, Leydig cells, Sertoli cells, and monocytes. Thedifferentiation from monocytes to macrophages is associated with adrastic Increase in LRP expression. Finally, LRP is expressed also incell types such as ape kidney cells (COS) or Chinese hamster ovary cells(CHO) (D. J. FitzGerald et al., J. Cell Biol. Vol. 129, 1995, pp.1533-1541), which are both frequently used to express mammalian proteinsincluding Factor VIII (R. J. Kaufman et al., Blood Coag. Fibrinol. Vol.8 (Suppl. 2), 1997, pp. 3-14).

LRP is involved in the clearance of a diversity of ligands includingproteases, inhibitors of the Kunitz type, protease serpin complexes,Upases and lipoproteins, which suggests that LRP plays an essential rolein various physiological and pathophysiological clearance processes(Narita et al., Blood, Vol. 2, pp. 555-560, 1998; Orth et al., Proc.Natl. Acad. Sci., Vol. 89, pp. 7422-7426, 1992; Kounnas et al., J. Biol.Chem., Vol. 271, pp. 6523-6529, 1996). LRP's physiological importancegoes back to the finding that LRP knock-out mice do not survive theembryonic stage (Herz, J. Curr. Opin. Lipidol. Vol. 4, 1993, pp.107-113). LRP secretion may be complicated by LRP interacting withmultiple ligands. Within the cell, LRP is, however, associated with itschaperone protein, the receptor-associated protein (RAP). If bound toRAP, LRP cannot interact with any of its known ligands (Herz et al., J.Biol. Chem., Vol. 266, pp. 21232-21238, 1991).

The interaction of LRP with its natural ligands may be effectivelyblocked by soluble LRP fragments. These fragments may be obtained byvarious methods known in the art, including recombinant techniques, andas such provide access to effective LRP antagonists (I. R. Horn, J.Biol. Chem., Vol. 272, 1997, pp. 13608-13613; B. Vash et al., Blood,Vol. 92, 1998, pp. 3277-3285).

In view of the typical role of LRP in the clearance of proteases,inhibitors and protease inhibitor complexes, it is to be noted that LRPalso binds the activated non-enzymatic cofactor Factor VIIIa (A.Yakhyaev et al., Blood Vol. 90 (Suppl. 1), 1997, 126-1 (Abstract)).While that disclosure suggests LRP's role in the regulation of FactorVIIIa, it does not give any hint as to its role in the regulation ofnon-activated heterodimeric Factor VIII, although this would be ofpotential interest for the clearance of Factor VIII from the bloodcirculation—and hence the half-life of Factor VIII.

There have been several prior art attempts to enhance thepharmacokinetic profile of Factor VIII, including modifications invarious regions of Factor VIII polypeptides.

WO 87/07144 describes various modifications of proteolytic interfacescomprising arginine and lysine residues, reducing the instability of themolecules for a specific protease-catalyzed cleavage, for instance theFactor VIIIa interface between Arg 1721 and Ala 1722.

WO 95/18827, WO 95/18828 and WO 95/18829 describe Factor VIIIderivatives with modifications in the A2 region of the heavy chain.

WO 97/03193 discloses Factor VIII polypeptide analogs in which themodifications comprise alterations of the metal binding properties ofthe molecule.

WO 97/03195 describes Factor VIII:C polypeptide analogs in whichmodifications are provided on one or several amino acid residuesadjacent an Arg residue.

EP-0 808 901 describes the construction of Factor VIII variantsincluding at least one mutation in at least one immunodominant region ofFactor VIII and the use of these Factor VIII variants in the treatmentof patients with Factor VIII inhibitors. Those modifications do notresult in an extended half-life or enhanced stability of the Factor VIIIvariant, neither in vivo nor in vitro.

In light of the prior art, none of the documents suggests that amodification in the light chain of Factor VIII will lead to a modifiedbinding affinity relative to a cell receptor and, consequently, to areduced clearance of the Factor VIII protein and an extended half-lifeand an enhanced stability of Factor VIII.

BRIEF SUMMARY OF THE INVENTION

Therefore, it is an object of the present invention to provide a FactorVIII polypeptide having Factor VIH:C activity, which exhibits anextended half-life and/or an enhanced stability of the Factor VIIIprotein in vivo and/or in vitro. Accordingly, the present inventionprovides a Factor VIII polypeptide containing a modification in thelight chain of the molecule, which influences the binding affinity tolow density lipoprotein receptor protein (LRP).

In a preferred embodiment of the invention, the Factor VIII polypeptidemodification is contained in the A3 domain, between amino acid sequence(AS) 1690 and 2032, in the C1 domain, between AS 2033 and 2172, and/orin the C2 domain of the light chain, between AS 2173 and 2332 (all aminoacid numerations made in the instant application in respect to theFactor VIII sequence refer to the numeration of Vehar et al. (Nature,Vol: 312, 1984, pp. 337-342), the entire contents of which are herebyincorporated by reference.

The modification in the A3 domain is contained, in particular, betweenAS 1722 (Met) and 1725 (Gly), AS 1743 (Phe) and 1749 (Arg), AS 1888(Ser) and 1919 (His), As 1942 (Trp) and 1947 (Met) and/or AS 1959 (Ser)and 1974 (Ala).

In another embodiment of the present invention, the modification in theC1 domain is contained between AS 2037 (He) and 2062 (Trp), AS 2108(Asp) and 2118 (Asn) and/or AS 2154 (Thr) and 2158 (He). In a morepreferred manner, the modification is contained between AS 2112 (Trp)and 2115 (Tyr).

Preferably, the modification is contained between AS 2209 (Arg) and 2234(Phe) and/or AS 2269 (His) and 2281 (Lys) of the C2 domain.

In an even more preferred manner, the modification is contained betweenAS 2211 (His) and 2230 (Leu).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the interaction between Factor VIII (Table A),thrombin-activated Factor VIII (Factor VIIIa, Table B), the heavy chainof Factor VIII (Table C) or the light chain of Factor VIII. (Table D),respectively, and immobilized LRP using surface plasmon resonanceanalysis. Details are provided in Example I. A comparison of Tables A-Dshows that Factor VIII, thrombin-activated Factor VIII, the light chainof Factor VIII, but not the heavy chain of Factor VIII, interactefficiently with LRP.

FIG. 2 shows that the light chain of Factor VIII binds to immobilizedLRP in a reversible and dose-dependent manner. The kinetic parameters ofthis interaction are summarized in Table II, which is set forth inExample II. The binding was judged as described in Example II.

FIG. 3 illustrates the action of the LRP antagonist RAP on theconcentration of the light chain of Factor VIII in a medium of cellsexpressing the light chain of Factor VIII. Assays were carried out asdescribed in Example III. In the absence of RAP (open symbols), theincrease in the light chain of Factor VIII within the medium is smallerthan in the presence thereof (closed symbols). Data illustratesexpression of the light chain of FVIII in CHO cells.

FIG. 4 illustrates the action of the LRP antagonist RAP on theconcentration of the intact Factor VIII heterodimer in the medium of theFactor-VIII-expressing cells. The details of the assay are described inExample IV. In the absence of RAP (open symbols), the increase in theFactor VIII activity is smaller than in the presence thereof (closedsymbols). Data illustrates expression of FVIII-dB685 in C127-cells.

FIGS. 5A and 5B illustrate hydropathy plots of the domains A3, C1 and C2of the light chain of Factor VIII. The plot was established as describedin Example VI. The plot shows the presence of different individualregions of low hydropathy values indicating the hydrophilic nature ofpotentially exposed exosites. They are shown as A to K (FIG. 5A) and Ito IV (FIG. 5B).

FIG. 6 illustrates the interaction of the C2 domain of Factor VIII withimmobilized LRP in the presence of the C2-domain-directed antibodyESH-8. Binding was analyzed using surface plasmon resonance as describedin Example VII. In the absence of the C2 domain, ESH-8 does not show anysignificant binding to immobilized LRP. In the presence of the C2domain, a dose-dependent increase in the binding to LRP is, however,observed. This demonstrates that the C2 domain of Factor VIII binds toLRP.

FIG. 7 illustrates the binding of the C2 domain of Factor VIII to LRP inthe presence of ESH4.

FIG. 8 illustrates the effect of RAP on the expression of FactorVIII-A3-C1 in CHO-K1 cells.

DETAILED DESCRIPTION

Within the context of the present invention, it has been found that theinhibition of LRP by its antagonist, RAP, results in Factor VIII lightchain accumulation in the medium. This proves that the cellularabsorption of the Factor VIII heterodimer encompasses an LRP-dependentmechanism.

Surprisingly, it has been shown that a modification in the light chainof the Factor VIM polypeptide has a similar effect, i.e., an increasedhalf-life and stability of the Factor VIII protein. Due to Factor VIIImolecule modification, the binding affinity to LRP decreases, whichinhibits the rapid clearance of the protein. This finding offers newoptions for an enhanced treatment of coagulation disturbances, whichmight be necessary in the preparation of Factor VIII compositions.

Due to the modification contained in the Factor VIII polypeptide, theincrease in the in vivo and in vitro half-lives of the Factor VIIImolecule according to the present invention may be at least 10%,preferably 25%, more preferably 60%, still more preferably 90%, ascompared to the wild-type Factor VIII protein.

Factor VIII polypeptides or Factor VIII variants made in accordance withthe teachings of the present invention exert their beneficial effectsbecause they constitute interactive regions (exosites) located on thesubunits of the Factor VIII heterodimer, in particular, on the lightchain of Factor VIII (domains A3-C1-C2). The term exosite is used hereinin its broadest sense, relating to relatively hydrophilic moieties ofthe protein, which are directed primarily at the surface of the FactorVIII molecule (Kyte and Doolittle, J. Mol. Bio!., Vol. 57, pp. 105-132,1982).

Although the process of Kyte and Doolittle operates according toprinciples already acknowledged in this field, based on the Factor VIIIsequence as previously published, no attention has so far beenpractically paid to these hydrophilic exosites. The exosite at aminoacid sequence (AS) Ser 1784 to Asp 1831, for instance, includes thebinding region of Factor IX, which has already been described in theliterature (AS 1801 to 1823, P. J. Lenting et al., J. Biol. Chem., Vol.271, pp. 1935-1940). This clearly demonstrates the relevance of thehydropathy plots used to identify exosites. The term “binding site”herein refers to a typical sequence pattern of amino acids, includingtheir natural and synthetic analogs which meet the minimum requirementsfor the binding of non-activated Factor VIII to LRP.

In a first group of preferred embodiments of the invention, thepolypeptide contains a modifcation in one or several of the exositeswithin the sequence of the Factor VIII polypeptide, preferably of thelight chain of Factor VIII, and more preferably of the C2 domain ofFactor VIII. In addition, these polypeptides preferably are derived fromthe sequence of human Factor VIII, although the invention comprisesbinding sites that are based on Factor VIII exosites of any desiredmammalian species.

Modification may be carried out for instance, by directed in vitromutagenesis, PCR, or other prior art methods of bioengineering suitablefor the specific alteration of a DNA sequence aimed at the directedreplacement of amino acids (Current Protocols in Molecular Biology, Vol.1, Chapt. 8 (Ausubel et al., Eds., J. Wiley and Sons, 1989 & Suppl.1990-93); Protein Engineering (Oxender & Fox Eds., A. Liss, Inc.,1987)). This modification may be comprised of a mutation, deletion, orinsertion in the Factor VIII light chain region.

Furthermore, the present invention provides the nucleic acids thatencodes each of the modified Factor VIII proteins encompassed by thepresent invention. The nucleic acids may be DNA or RNA. The nucleicacids are contained in an expression vector that provides the elementswhich are suitable for the expression of this DNA or RNA. For instance,the expression vector may comprise, in the transcription direction, atranscriptional regulation region and a translational initiation regionwhich are functional in a host cell, a DNA sequence encoding the FVIIIpolynucleotide of the present invention, and translational andtranscriptional termination regions that are functional in this hostcell, and the expression of this nucleic sequence being regulated by theinitiation and termination regions. The expression vector also maycontain elements for the replication of this DNA or RNA. The expressionvector may be a DNA or RNA vector. Examples of DNA expression vectorsinclude pBPV, pSVL, pRc/CMV, pRc/RSV, myogenic vector systems asdisclosed in (WO 93/09236) or vectors originating from virus systems,for instance, from vaccinia virus, adenoviruses, adeno-associated virus,herpes viruses, retroviruses or baculoviruses. Examples of RNAexpression vectors include vectors originating from RNA viruses such asretroviruses or flaviviruses.

The nucleic acids, DNA, and RNA may be chemically modified for thosespecific applications in genetic therapy where nucleic acids areinjected into the organ of a mammal. Chemical modifications may includemodifications to protect the nucleic acid against nuclease digestion,for instance, by stabilizing its skeleton or termini.

The expression vector which contains the nucleic acid encoding themodified Factor VIII polypeptide according to the present invention maybe used to transform host cells which will then produce thispolypeptide. The transformed host cells may be grown in a cell culturesystem in order to produce this polypeptide in vitro. The host cells cansegregate the modified Factor VIII polypeptide into the cell culturemedium, from which it can be purified. The host cells also can keep themodified Factor VIII polypeptide within their cell walls, and the hybridprotein may be produced from the host cells.

Mammalian body cells such as fibroblasts, keratinocytes, hematopoieticcells, hepatocytes, or myoblasts may be used as host cells. The hostcells are transformed in vitro by an expression vector system thatcarries a nucleic acid made in accordance with the teachings of thepresent invention and are reimplanted into the mammal. The Factor VIIIpolypeptide encoded by this nucleic acid is synthesized in vivo by thesecells, and they will exhibit a desired biological activity in themammal. According to one embodiment of the present invention, the mammalis a human patient suffering from hemophilia.

The nucleic acid sequence encoding the modified Factor VIII polypeptidemade in accordance with the teachings of the present invention also maybe used to create transgenic animals expressing these modified FactorVIII polypeptide proteins in vivo. In one embodiment of this specificapplication, the transgenic animals are able to produce the Factor VIIIpolypeptide in endogenous glands such as mammary glands, from whichthese proteins can be separated. For example, Factor VIII proteinsproduced in the mammary glands can be separated into the milk of theanimals to produce these proteins/The animals may include, but are notlimited to, mice, cattle, pigs, goats, sheep, rabbits or any othereconomically useful animal.

Furthermore, the expression vector which contains the nucleic acidencoding for any Factor VIII polypeptide encompassed by the presentinvention may be administered to mammals without previous in vitrotransformation in host cells. The practical background for this type ofgenetic therapy is disclosed in several patent applications such as WO90/11092. The expression vector containing this nucleic acid sequence ismixed with a suitable carrier, such as a physiological buffer solution,and injected into an organ, preferably a skeletal muscle, the skin, orthe liver of a mammal. The mammal preferably is a human being and, morepreferably, is a subject suffering from a genetic defect and, mostpreferably, a subject suffering from a blood coagulation disturbance. Ina particular embodiment, the mammal is a human patient suffering fromhemophilia, and the nucleic acid contained in the expression vectorencodes the modified Factor VIII polypeptide, as described.

It is advantageous that the modified Factor VIII protein according tothe present invention has a Factor VIII procoagulant activity of atleast 50%, more preferably at least 80%, in particular at least 100%, ofthe Factor VIII procoagulant activity of a Factor VIII protein withoutthe modification that leads to a reduced binding affinity to LRP, forinstance, of a commercially available Factor VIII preparation based onrecombinant or plasmatic Factor VIII:C.

The evaluation of the Factor VIII procoagulant activity may be effectedby means of any suitable test, in particular those tests which areroutinely carried out in the investigation of Factor VIII samples suchas the one-stage clot test as described in Mikaelsson and Oswaldson,Scand. J. Haematol. Suppl. 33, pp. 79-86, 1984, or a chromogenic testsuch as Factor VIII IMMUNOCHROM (Immuno).

The Factor VIII activity also may be determined by measuring thecapability of Factor VIII to function as a cofactor to Factor IXa in theconversion of Factor X to Factor Xa, using a chromogenic substrate forFactor Xa (Coatest Factor VIII, Chromogenix, Moelndal, Sweden). Inaddition, other tests that serve to determine the amount of Factor VIIIactivity in a sample may be used to test the Factor VIII activity of themodified proteins described in the present invention.

The actual test whether any of the newly modified Factor VIII proteinsexhibits a defined percentage of Factor VIII procoagulant activity ispreferably carried out in parallel with a test on the same Factor VIIImolecule without modification in the LRP binding domain (e.g., FactorVIII wild type or a fully active Factor VIII with a deleted B domain). Acalibrated test of the mutant Factor VIII molecule enables theexamination of the relative procoagulant activity (the percentage of theactivity as compared to a 100% activity of the wild type or of FactorVIII including a B domain deletion) without the risk of an error onaccount of medium factors. Since the results of in vitro tests forFactor VIII procoagulant activity are often influenced by errors thatare due to the artificial nature of the same, the two propertiespreferably are assayed also by in vivo or ex vivo tests in order toobtain more reliable results in respect to activity values.

Like the in vitro tests, parallel testing of the Factor VIII moleculewithout modification is also preferred if in vivo tests are carried out.Animal models suitable for the evaluation of the Factor VIII:C activityare described in WO 95/01570 and EP 0 747 060.

A preparation made in accordance with the teachings of the presentinvention may be provided as a pharmaceutical preparation comprising amodified Factor VIII polypeptide either as a single-componentpreparation or combined with other components, as a multi-componentsystem. In one embodiment, the Factor VIII proteins or the modifiedFactor VIII molecules made in accordance with the teachings of thepresent invention may be combined with one or several polypeptides suchas RAP that selectively inhibit the binding and internalization ofFactor VIII by low density lipoprotein receptor-related proteins (LRP).

The present invention also contemplates a composition comprising aFactor VIII molecule and one or several polypeptides antagonisticallyinterfering with the interaction between Factor VIII and LRP resultingin the selective inhibition of the binding and internalization of FactorVIII by LRP. Preferably, the polypeptide is RAP or a soluble LRPfragment having an antagonistic effect. Preferably, the soluble LRPfragment binds to Factor VIM in the Factor VIII-LRP binding region.

These preparations may be used as active components of pharmaceuticalcompositions for the treatment of patients suffering from geneticdisturbances, preferably coagulation disturbances, and most preferablyhemophilia, for instance hemophilia A. Moreover, these compounds may beused as active components of pharmaceutical compositions for thetreatment of patients suffering from temporary disturbances of theirthrombotic or fibrinolytic systems that may occur before, during, orafter an operation.

In accordance with the present invention, a pharmaceutical compositionis intended for the treatment to mammals, preferably humans. Whenproducing the pharmaceutical product of the present invention, compoundsof the present invention, modified Factor VIII polypeptides, nucleicacids encoding the same, or the transformed cells capable of in vivoexpression of Factor VIII polypeptides are mixed with physiologicallyacceptable carriers.

The compositions disclosed in the present invention may be formulatedfor administration in any suitable way, and the invention alsoencompasses pharmaceutical compositions containing a therapeuticallyeffective amount of Factor VIII. The compositions of the presentinvention may be formulated in a conventional manner using one orseveral pharmaceutically acceptable carriers or excipients. Suitablecarriers include, but are not limited to, diluents or fillers, sterileaqueous media and various nontoxic organic solvents. The compositionsmay be formulated in the form of powders, aqueous suspensions orsolutions, injectable solutions and the like. Suitable dosage forms willbe readily identified by the skilled artisan.

According to the methods of the present invention, treatments forcoagulation disturbances should be carried out using a dosage schemethat will guarantee the maximum therapeutic response until improvementhas been reached and a subsequent effective minimum dosage amount thatoffers a suitable protection against bleeding. The dosage forintravenous administration may range between about 10 and 300 IU/kg bodyweight, preferably between about 10 and 100 IU/kg body weight, and morepreferably between 20 and 40 IU/kg body weight. A suitable dosage mayalso depend on the patient's age, general health, or other factors thatmay influence the response to the drug. The drug may be administered bycontinuous infusion or at regular intervals in order to keep thetherapeutic effect on the desired level.

Another aspect of the invention relates to a method for producingmodified Factor VIII molecules, which contain a modification in thelight chain. The sequence encoding the modified Factor VIII molecule isinserted in a suitable expression system such as an expression vector,and suitable cells are transfected with the recombinant DNA. Preferably,permanent cell lines expressing the modified Factor VIII areestablished. The cells are grown under conditions that are optimal forgene expression, and modified Factor VIII is isolated, either from acell culture extract or from the cell culture supernatant. Therecombinant molecule may be further purified by means of any knownchromatographic methods such as anion or cation exchange chromatography,affinity or immunoaffinity chromatography, or a combination thereof.

Modified Factor VIII is preferably produced by recombinant expression.It may be produced recombinantly by means of any usual expression systemsuch as, but not limited to, permanent cell lines or viral expressionsystems. Permanent cell lines are produced by the stable integration offoreign DNA into the host cell genome of, for instance, vero, MRC5, CHO(Chinese Hamster Ovary), BHK (baby hamster kidney), 293, Sk-Hep1 cells,in particular hepatic and renal cells, fibroblasts, keratinocytes ormyoblasts, hepatocytes or stem cells, hematopoietic stem cells, or by anepisomal vector derived, for instance, from papilloma virus. Virusexpression systems such as vaccinia virus, baculovirus or retrovirussystems may likewise be used. Generally, vero, MRC5, CHO, BHK, 293,Sk-Hep1, glandular, hepatic and renal cells are used as cell lines.Eukaryotic expression systems that may be used include yeast cells,endogenous glandular cells (e.g., glands of transgenic animals) and alsoother types of cells. Naturally, transgenic animals may also be used forthe expression of the polypeptides of the present invention orderivatives thereof. CHO-DHFR cells have proved to be particularlysuitable for the expression of recombinant proteins (Urlaub et al.,Proc. Natl. Acad. Sci., U.S.A., Vol. 77, pp. 4216-4220, 1980).

Prokaryotic expression systems may also be used for the recombinantproduction of modified Factor VIII made in accordance with the teachingsof the present invention. Systems enabling an expression in E. coli orB. subtilis are particularly suited.

The Factor VIII polypeptide of the present invention is expressed in therespective expression system under the control of a suitable promoter.Any of the known promoters such as SV40, CMV (cytomegalovirus), RSV(respiratory syncytial virus), HSV (herpes simplex virus), EBV (EpsteinBarr virus), p-actin, hGH (human growth hormone) or inducible promoterssuch as, e.g., hsp or metallothionein promoter are suitable foreukaryotes expression.

According to the present invention, a total-length Factor VIII-cDNA aswell as any of its derivatives comprising Factor VIII:C activity (forinstance, B-domain-deleted Factor VIII mutants, Factor VIII mutantsincluding partially deleted B domains) may be used as starting materialsfor the construction of the modified Factor VIII polypeptide. It may bederived from any mammalian species, preferably human, swine or bovinesources.

The present invention is illustrated in the examples described below.Although illustrative of the present invention in respect to theidentification, production and use of enhanced compositions with areduced binding to LRP of the light chain of Factor VIII, the presentinvention also should be interpreted to be applicable to the LRP bindingof the heavy chain of Factor VIII. Variations known by those of ordinaryskill in the art are to be regarded as falling within the scope of thepresent invention. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as generallyunderstood by the average skilled artisan in the field to which thepresent invention pertains. Although any methods and materials similaror equivalent to those described herein may be employed in practising ortesting the present invention, preferred methods and materials will nowbe described.

EXAMPLES Example I The Light Chain of Factor VIII has an LRP BindingSite

The binding of Factor VIII and subunits of the same to low densitylipoprotein receptor-related protein (LRP) was examined using purifiedcomponents. LRP, Factor VIII, the light chain of Factor VIII, the heavychain of Factor VIII, and thrombin-activated Factor VIII were obtainedusing established methods (Moestrup S. K. et al._(t) J. Biol. Chem.,Vol. 266, 1991, pp. 14011-14017; Lenting P. J. et a!., J. Biol. Chem.,Vol. 269, 1994, pp. 7150-7155; and Curtis J. E. et al., J. Biol. Chem.,Vol. 269, 1994, pp. 6246-6250, respectively).

The interaction with LRP was examined on a BIAcore™2000 Biosensor System(Pharmacia Biosensor AB, Uppsala, Sweden) using surface plasmonresonance (SPR) analysis. LRP was immobilized on a CM5 sensor chip at aconcentration of 8.3 fmol/mm², the amine coupling set having been usedaccording to the manufacturer's instructions (Pharmacia Biosensor,Uppsala, Sweden). A control channel on the sensor chip was activated andblocked, using amine coupling reagents without protein immobilization.

Factor VIII or derivatives thereof were passed over the control channelat a concentration of 100 nM in order to assess non-specific binding,and over the LRP-coated channel in 50 mM HEPES (pH 7.4), 150 mM NaCl, 2mM CaCl₂ and 0.005% (v/v) Tween 20 at a flow of 5 ml/min for a period of2 min at 25° C. The association between the different proteins and LRPis indicated in FIG. 1 and expressed in resonance units. In Table I, themaximum increase in the resonance units for the different derivatives issummarized. The data demonstrated that Factor VIII, thrombin-activatedFactor VIII and the light chain of Factor VIII are able to interact withLRP. In contrast, the heavy chain of Factor VIII could not bind LRP.Therefore, it is apparent that the binding moiety of Factor VIII orthrombin-activated Factor VIII for LRP is located in the A3-C1-C2 region(residues 1690-2232).

TABLE I Binding of Factor VIII and its subunits to immobilized LRP asdetected by SPR analysis. Binding to LRP is expressed in resonance unitsand has been corrected in regard to nonspecific binding. Protein Binding(resonance units) Factor VIII 262 Heavy chain of Factor VIII 0 Lightchain of Factor VIII 305 Thrombin-activated Factor VIII 446

Example II Association Kinetics of Immobilized LRP and the Light Chainof Factor VIII

The kinetic parameters for the interaction between the light chain ofFactor VIII and immobilized LRP were determined on a BIAcore™2000Biosensor System (Pharmacia Biosensor AB, Uppsala, Sweden) using SPRanalysis. This method is generally known in the art and was employed forthe kinetic analysis of the interaction between LRP andreceptor-associated protein (RAP) (Horn I, in LRP-ligand interactions:kinetics and structural requirements; Ph.D. thesis, 1997, pp. 65-106,Amsterdam University). LRP was immobilized at a concentration of 6.7fmol/mm² on three channels of a CM5 sensor chip as described in ExampleI. A control channel used to evaluate nonspecific binding was preparedas described in Example I. Different concentrations of the light chainof Factor VIII (150, 175, 200, 225 and 250 nM) were passed over thecontrol channel and over the LRP-coated channel in 50 mM HEPES (pH 7.4),150 mM NaCl, 2 mM CaCl2 and 0.005% (v/v) Tween 20 at a flow of 20 ml/minfor a period of 2 min at 25° C. so as to enable association.Subsequently, the channels were incubated with the same buffer at asimilar flow so as to enable dissociation. As depicted in FIG. 2, adose-dependent association and dissociation curve is observed.

The data were analyzed by means of Biacore Evaluation Software(Pharmacia Biosensor AB, Uppsala, Sweden). The data analysisdemonstrated that the interaction between the light chain of Factor VIIIcorresponded best with two classes of binding sites. The association anddissociation rate constants (k_(on) and k_(off), respectively) werecalculated for the two binding sites. These speed constants weresubsequently used to obtain the equilibrium constants (K_(d)) for theseinteractions.

TABLE II Speed constants for the interaction between the light chain ofFactor VIII and immobilized LRP. The data analysis indicates theinteraction of the light chain of Factor VIII with two classes ofbindings sites represented by A and B, respectively. Class kon (M−¹s*¹)ko_(ff) (s−¹) Kd (=Koff/ko_(n); nM) A 3.0 × 10⁵ 5.5 × 10′² 182 B 7.2 ×10⁴ 2.7 × 10″³ 37

Example III Interaction Between the Light Chain of Factor VIII andCell-Bound LRP

Since the light chain of Factor VIII binds effectively to LRP in asystem in which purified components are used, the interaction betweenthe light chain of Factor VIII and LRP expressed on the surface ofliving cells was investigated. In order to express the light chain ofFactor VIII, i.e., residues 1649 to 2332 (Toole J. J. et al., Nature,Vol. 312, 1984, pp. 342-347), a construct encoding the Factor VIIIsignal peptide fused to residues 1649 to 2332 was made. This constructwas prepared by using the previously described plasmid pBPV-FactorVIII-dB695 (K. Mertens et al., Brit. J. Haematol., Vol. 85, 1993, pp.133-142) as a template for the preparation of two Factor VIII fragmentsusing polymerase chain reaction (PCR). A fragment was made using thesense primer A1 (5′-TTA GGA TCC ACC ACT ATG CAA ATA GAG CTC TCC-3¹) (SEQID NO: 1), which contained a BamH1 recognition site and a moietyencoding the N-terminal residues of the Factor VIII signal peptide, andthe antisense primer A1 (5′-AGT AGT ACG AGT TAT TTC ACT AAA GCA GAA TCGC-3′) (SEQ ID NO: 2) encoding C-terminal residues of the Factor VIIIsignal peptide and N-terminal residues of the light chain of FactorVIII. A second fragment was made using the sense primer B1 (5′-TTG CGATTC TGC TTT AGT GAA ATA ACT CGT ACT AC-3¹) (SEQ ID NO: 3) encoding theC-terminal residues of the Factor VIII signal peptide and the N-terminalresidues of the light chain of Factor VIII, and the antisense primer B1(5′-ATT GCG GCC GCT CAG TAG AGG TCC TGT GCC TC-3¹) (SEQ ID NO: 4)encoding a Not1 recognition site, a stop codon and a moiety encoding theC-terminal residues of the light chain of Factor VIII.

In a second reaction, the products of the two reactions were used as atemplates for the construction of the resulting fragment referred to asFactor VIII-SPLC, using primers A1 and B1. Factor VIII-SPLC consisted ofa BamH1 recognition site, a moiety encoding the Factor VIII signalpeptide and fused to a moiety encoding the light chain of Factor VIII, astop codon and a Not1 recognition site. Factor VIII-SPLC subsequentlywas digested with BamH1 and Not1 and ligated into the expression vectorpcDNA3, 1 (Invitrogen, Leek, the Netherlands), which was digested usingthe same restriction enzymes. The resulting vector with the designationpcFactor-VIII-LC was transfected into Chinese hamster ovary K1 (CHO-K1)cells (ATCC CCL-61) using calcium phosphate precipitation (J. Sambrooket al., Molecular Cloning; A Laboratory Manual, Cold Spring LaboratoryPress, Cold Spring Harbor, U.S.A., 1989, p. 1637). CHO-K1 cells wereestablished to express LRP. constitutively on its cell surface (D. J.FitzGerald et al., J. Cell. Biol., Vol. 129, 1995, pp. 1533-1541).Stably expressing CHO-K1 cells were obtained at a concentration of 800μg/ml upon selection with G-148 (Gibco-BRL, Breda, the Netherlands).

CHO-K1 cells stably expressing the light chain of Factor VIII were grownto confluence in 2 wells of a 6-well plate (Nunc A/S, Roskilde,Denmark). The wells were washed five times using Dulbecco's modifiedEagle medium F12 (DMEM-F12) (Gibco, BRL, Breda, the Netherlands) and 1ml DMEM-F12 was added. In one well, the LRP antagonist, RAP, was addedimmediately to a concentration of 20 mg/ml at 2 and 4 hours after cellwashing. Samples were drawn up to six hours after cell washing and thenanalyzed for the concentration of the light chain of Factor VIII using amethod known in the art (Lenting P. J. et al., J. Biol. Chem., Vol. 269,1994, pp. 7150-7155). As illustrated in FIG. 3, the concentration of thelight chain of Factor VIII in the medium increased with time in theabsence of RAP. However, in the presence of RAP, the extent of theincrease of the light chain of Factor VIII rose as compared to theabsence of RAP. Thus, the inhibition of LRP is associated with anaccumulation of the light chain of Factor VIII in the medium. Thisclearly demonstrates that an LRP-dependent mechanism is involved in thecellular uptake of the light chain of Factor VIII.

Example IV Interaction, Between Factor VIII and Cell-Surface-Exposed LowDensity Lipoprotein Receptor-Related Protein

As described in Example III, an interaction occurs between the lightchain of Factor VIII and the cell-surface-exposed LRP. Therefore, alsoexamined was whether the intact Factor VIII protein interacts withcell-surface-exposed LRP. A previously established, mouse fibroblastcell line which was stably transfected in order to produce Factor VIII(Mertens K. et al., Brit. J. Haematol., Vol. 85, 1993, 133-142) wasgrown to confluence in 2 wells of a 6-well plate (Nunc A/S, Roskilde,Denmark). The cells were washed five times using Iscov's modified Eaglemedium (IMEM) (Boehringer Ingelheim/Biowhitaker, Verviers, Belgium), and1 ml IMEM was added. In one well, LRP antagonist, RAP, was addedimmediately, to a concentration of 20 mg/ml at 2 and 4 hours after cellwashing. Samples were drawn up to six hours after cell washing and thenanalyzed for Factor VIII-cofactor activity using an already establishedmethod (Mertens K. et al., Brit. J. Haematol., Vol. 85, 1993, 133-142).As shown in FIG. 4, the amount of Factor VIII-cofactor activity in themedium increases with time in the absence of RAP. However, in thepresence of RAP, the extent of the increase of Factor VIII rose ascompared to the absence of RAP. Thus, the inhibition of LRP isassociated with an accumulation of Factor VIII in the medium. Therefore,it is apparent that an LRP-dependent mechanism is involved in thecellular uptake of the light chain of Factor VIII.

Example V The Action of RAP on the Factor VIII Pharmacokinetics inKnock-Out Mice Suffering from Severe Factor VIII Deficiency

A mouse strain suffering from severe Factor VIII (FVIII) deficiency wasrecombinantly created by the selective disruption of the mouse FactorVIII gene according to Bi et al., Nature Genetics, 1995, Vol. 10, pp.119-121. Factor VIII knock-out mice were created by inserting a neo-geneinto the 3¹ end of exon 17 of the mouse Factor VIII gene. The affectedmale animals (X″Y) had nondetectable Factor VIII levels of <0.02±0.01U/ml when measurements were carried out either by a chromogenic FactorVIII test, Hyland Immuno, Vienna, Austria, as recently described(Turecek et al., Thromb. Haemostas. Suppl., 1997, Vol. 769) or byantigen ELISA as described below.

Two affected hemizygous male mice (X′Y) were intravenously treated witha dose of 200 U/kg body weight of a recombinant human Factor VIII(rhFVIII) preparation which was derived from Chinese hamster ovary cellsproduced as described (WO/85/01961) and pharmaceutically formulatedwithout stabilizing protein.

One hour after treatment, the tips of the tails of the narcotized micewere incised by the edge of a scalpel as described by Novak et al.,Brit. J. Haematol. Vol. 69, 1998, pp. 371-378. A volume of 50 μl bloodwas collected from the tail wounds by means of capillary tubes(Ringcaps, Hirschmann, Germany), which capillary tube were coated withlithium heparin as an anticoagulants. The capillary tubes were closedand centrifuged to separate blood cells and plasma. The capillary tubeswere opened, and the cell and plasma fractions were collected by furthercentrifugation. Finally, the plasma samples were subjected to FactorVIII determination by means of Factor VIM antigen ELISA, test setIMMUNOZYM FV1I1 Ag, Hyland Immuno, Vienna, Austria, using monoclonalanti-Factor-VIII-antibodies both for capturing and for detection asdescribed in Stel et al., Nature, 1983, Vol. 303, pp. 530-532; Lentinget al., J. Biol. Chem., Vol. 269, 1994, pp. 7150-7155; Leyte et al.,Biochem. J., Vol. 263, 1989, pp. 187-194. The resulting Factor VIIIvalues were expressed in International Units of human Factor VIII. Theresults of the Factor VIII plasma levels are indicated in the Table.

Two other affected hemizygous male mice (X′Y) were pretreated withrecombinant receptor-associated protein (GST-RAP) 10 minutes prior tothe treatment with recombinant human Factor VIII at a dose of 40 mg/kgbody weight. The RAP used in this assay, which interacts with LRP, wasobtained by bacterial fermentation as described by Hertz et al. (J.Biol. Chem., Vol. 266, 1991, pp. 21232-21238). A fusion protein of RAPwith glutathion-S transferase was expressed in E. coli and purified byaffinity chromatography on glutathione agarose. The resulting proteinprimarily consisted of the fusion protein and cleavage products of RAPand glutathione-S transferase. The fusion protein was formulated in aninjectable buffer ready for administration to the Factor VIII knock-outmice. As in the control group (treatment solely with Factor VIII), bloodsamples were drawn one hour after the administration of recombinantFactor VIII and measured for their Factor VIII activity using FactorVIII antigen ELISA. The results are indicated in Table III.

TABLE III Recovery Mouse Treatment Dose Treatment Dose 1 h aftertreatment no. GST-RAP RhFVIII FVIII:Ag (U/ml plasma) 1 40 mg/kg 200 U/kg1.92 2 40 mg/kg 200 U/kg 1.88 3 — 200 U/kg 0.73 4 — 200 U/kg 0.83

In mice pretreated with GST-RAP, the Factor VIII level was more than200% of the plasma levels after treatment with recombinant Factor VIIIalone. The administration of the LRP antagonist, RAP, enhanced thepharmacokinetics of Factor VIII.

Example VI Identification of Potential LRP Binding Exosites on the LightChain of Factor VIII

A method for identifying exosites that may be involved in proteininteraction, has already been established (J. Kyte and R. F. Doolittle,J. Mol. Biol. Vol. 157, 1982, pp. 105-132). The method provides aprogram continuously evaluating the hydrophilicity and hydrophobicity ofa protein along its amino acid sequence. The method employs a hydropathyscale that indicates the average hydropathy within segments ofpredetermined sizes along the amino acid sequence. Hydrophilic sectionsare characterized by negative hydropathy values, and these sections areprobably oriented to the external side of a protein present in anaqueous solution. This method was applied to the known sequence of humanFactor VIM (G. A. Vehar et al., Nature, Vol. 312, 1984, pp. 337-342; J.J. Toole et al., Nature, Vol. 312, 1984, 342-347) using a segment size(“window”) of 19 residues. From the complete sequence of Factor VIII,the region 1690-2332 corresponding to the Factor VIII A3/C1/C2 domainwas subjected to this analysis, and the resulting hydropathy plot,having a cut-off value of −15, is illustrated in FIGS. 5A and 5B.

The results of this method show several isolated regions having lowhydropathy values, which reflect the hydrophilic nature associated withpotential exosites. The potential exosites are denoted by A to K (TableIV):

TABLE IV Site Residues Domain A Met 1711 to Gly 1725 A3 BPhe1743toArg1749 A3 C Ser 1784 to Asp 1831 A3 D Ser 1888 to His 1919 A3E Trp 1942 to Met 1947 A3 F Ser 1959 to Ala 1974 A3 G Ile 2037 to Trp2062 C1 H Asp 2108 to Asn 2118 C1 I Thr 2154 to Ile 2158 C1 J Arg 2209to Phe 2234 C2 K His 2269 to Lys 2281 C2

TABLE V Site Residues Domain I Phe 1785 to His 1822 A3 II Trp 1889 toAsn 1915 A3 III Trp 2112 to Tyr 2115 C1 IV His 2211 to Leu 2230 C2

From the complete sequence of Factor VIII, the region 1690-2332corresponding to the light chain of complete Factor VIII, was subjectedto this analysis, and the resulting hydropathy plot, which has a cut-offvalue of −20, is illustrated in FIG. 5B. The exosites are denoted by Ito IV.

Example VII The C2 Domain of Factor VIII Includes an LRP Binding Site

The A3-C1-C2 region of Factor VIII comprises the binding moiety for LRP(cf. Example I). This region contains a number of potential LRP bindingexosites in the domains constituting them, including the C2 domain (cf.Example VI). To demonstrate that such exosites might actually beinvolved in LRP binding, the interaction between LRP and the C2 domainof Factor VIII was analyzed more thoroughly. The C2 domain of FactorVIII (i.e., residues 2171-2332) was expressed in insect cells using anestablished method (K. Fijnvandraat et al., Blood, Vol. 91, 1998, pp.2347-2352). The C2 domain of Factor VIII was purified by immunoaffinitychromatography using the monoclonal antibody CLB-CAg 117 directed to theC2 domain (K. Fijnvandraat et al., Blood, Vol. 91, 1998, pp. 2347-2352).The interaction with LRP was assayed on a BIAcore™2000 System (PharmaciaBiosensor AB, Uppsala, Sweden) using surface plasmon resonance (SPR)analysis. LRP was immobilized on a CM5 sensor chip as described inExample I. In order to enhance the resonance signal, the C2 domain ofFactor VIII (0, 100 or 275 nM) was preincubated in the presence of 500nM of the monoclonal antibody ESH-8 directed at the C2 domain (D.Scandella et al., Blood, Vol. 86, 1995, pp. 1811-1819), in 50 mM HEPES(pH 7.4), 150 mM NaCl, 2 mM CaCl₂, 0.005% (v/v) Tween 20 for 15 min atroom temperature. The preincubated samples were then passed over thecontrol channel in order to assess nonspecific binding, as well as overthe LRP-coated channel (8.3 fmol/mm²), at a flow of 5 ml/min for 2 minat 25° C.

In the absence of the C2 domain, ESH-8—if at all—shows a minimum bindingto immobilized LRP. However, in the presence of the C2 domain, adose-dependent increase in the binding to LRP was observed (FIG. 6).This demonstrates that the C2 domain of Factor VIII binds to LRP. Thus,the exosites within the light chain of Factor VIII are definitelycapable of LRP binding and are involved in the LRP-dependent clearanceof Factor VIII in vivo.

It will be readily understood by the skilled artisan that the presentinvention is well apt to fulfill the tasks and achieve the mentioned aswell as inherent goals and advantages. The compounds, methods, andcompositions described herein are illustrated as representative of thepreferred embodiments. They are intended to exemplify the inventionwithout restricting its scope. Modifications and other uses are readilyconceivable by the skilled artisan and are intended to be encompassed bythe spirit of the invention and the scope of the annexed claims.

Example VIII Binding of the Factor VIII C2 Domain in the Presence of anAnti-Factor VIII-C2-Domain-Antibody

The Factor VIII (FVIII) molecule comprises two sites which are involvedin vWF binding, one of these sites being located on the carboxy-terminalC2 domain of the light chain of Factor VIII (Saenko and Scandella, J. B.C. 272 (1997), pp. 18007-18014). Since vWF binding is inhibited by theantibody ESH4 directed against the C2 domain, this effect was examinedfor LRP binding. The antibody binding body examinations having beencarried out as described in Example VII. The antibody ESH4 was obtainedfrom American Diagnostica.

As illustrated in FIG. 7, ESH4 interferes with the binding of LRP to thelight chain of Factor VIII. This inhibition appears to be specific,since ESH4 was not able to influence the binding of tissue-typeplasminogen activator/plasminogen activator inhibitor 1 complexes toLRP. Moreover, other antibodies which were directed against the lightchain of Factor VIII, i.e., CLB-CAg A and CLB-CAg 69 (Lenting et al., J.B. C. 269 (1994), pp. 7150-7155) were not able to interfere with LRP asregards the binding to Factor VIII.

FIG. 7 depicts the binding of the Factor VIII C2 domain to LRP in thepresence of ESH4. To this end, immobilized LRP (16 fmol/mm²) wasincubated with the light chain of Factor VIII (150 nM) in the presenceor absence of antibody ESH4 at a flow of 5 μl/min for 2 min at 25° C.The results were indicated in resonance units (RU) and corrected againstnonspecific binding, which was less than 5% as compared to the bindingto LRP-coated channels.

In FIG. 7, the concentration of ESH4 antibody in nM was plotted on theX-axis and the remaining binding of the light chain of Factor VIII in RUwas plotted on the Y-axis.

Example IX The Factor VIII A3-C1 Region Comprises an LRP Binding Site

The A3-C1-C2 region of Factor VIII comprises the binding moiety for LRP(cf. Example (I). From the kinetic analysis described in Example II, itis clearly apparent that multiple sites involved in LRP are present(Table II, class A and class B binding sites). As indicated in ExampleVII, the presence of such interactive sites for the region of the FactorVIII C2 domain was confirmed (cf. also FIG. 6). In order to confirm thatalso other exosites are involved in the interaction with LRP, theinteraction between LRP and the Factor VIII A3-C1 region (i.e., FactorVIM residues 1649 to 2172) was analyzed. In order to obtain the fragmentof the light chain of this Factor VIII, a construct encoding the FactorVIII signal peptide fused to residues 1649 to 2172 was prepared. Thisconstruct was prepared by the following method. The vector pcFactorVIII-LC described in Example III was used as a conformation template forthe construction of a Factor VIII fragment using PCR. This fragment wasproduced using the sense primer A1 (5′-TTA GGA TCC ACC ACT ATG CAA ATAGAG CTC TCC-3′) (SEQ ID NO: 1) and the antisense primer FA2172min(5′-AAT GCG GCC GCT TCA ATT TAA ATC ACA GCC CAT-3¹) (SEQ ID NO: 5). Theprimer FA 2172 encodes a Not1 recognition site, a stop codon and theresidues 2167 to 2172 of Factor VIII. The PCR product was cleaved withBspMII and NotI, and a 352 base pair fragment was isolated and ligatedinto the pcFactor VIII-LC vector, which was digested using the samerestriction enzymes. The resulting vector, which was designated aspcFactor VIII-A3C1, was transfected on CHO-K1 cells (ATCC CCL-61) usingcalcium phosphate precipitation (J. Sambrook et al., Molecular Cloning;A Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor,U.S.A., 1989, p. 1637). CHO-K1 cells which stably express Factor VIIIA3-C1 cells were obtained at a concentration of 800 ug/ml upon selectionwith G-148 (Gibco-BRL, Breda, the Netherlands).

CHO-K1 cells stably expressing Factor VIII A3-C1 fragments were used forlarge-scale cultivation in order to obtain a conditioned mediumcontaining Factor VIII A3-Cl. Factor VIII A3-C1 was purified byimmunoaffinity chromatography using the previously described monoclonalantibody CLB-CAg A (Leyte A. et al., Biochem. J., 1989, Vol. 263, pp.187-194) directed against the A3 domain of Factor VIII. To this end,CLB-CAg A was immobilized on CNBr Sepharose 4 B (Pharmacia Biotech,Roosendaal, the Netherlands) according to the manufacturer'sinstructions at a concentration of 1 mg/ml. A Conditioned medium wasincubated with CLB-CAg A Sepharose (2 ml per liter medium) and boundFactor VIIIA3-C1 was eluted in 150 mM NaCl, 55% (v/v) ethyleneglycol, 25mM lysine (pH 11). Factor VIII A3-C1 containing fractions wereimmediately neutralized using 1/10 volume of 1 M imidazole (pH 5.0) andthen dialyzed against 150 mM NaCl, 2 mM CaCl₂ and 0.005% (v/v) Tween 20,20 mM HEPES (pH 7.4).

The interaction between Factor VIII A3-C1 and LRP was investigated on aBIAcore™2000 Biosensor System (Pharmacia Biosensor AB, Uppsala, Sweden)using surface plasmon resonance analysis. LRP was immobilized on a CM5sensor chip as described in Example I. Samples that contained FactorVIII A3-C1 (200 nM or 400 nM) were passed over the control channel at aflow of 5 μl/min for a period of 2 min at 25° C. to assess nonspecificbinding and over the LRP-coated channel (8.3 fmol/mm²). In Table VI, themaximum increase in the resonance units for both concentrations ofFactor VIII A3-C1 is summarized. In the presence of 400 nM Factor VIIIA3-C1 a higher response was observed than with 200 nM Factor VIIIA3-C1(59 and 47 resonance units, respectively). In order to enhance thebinding of Factor VIII A3-C1 to LRP, Factor VIII A3-C1 (400 nM) waspreincubated in the presence of 500 nM of the monoclonal antibodyCLB-CAg A in 50 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM CaCl₂, 0.005% (v/v)TWEEN 20 for 15 min at room temperature. The preincubated samples werethen passed over the control channel and over the LRP-coated channel ata flow of 5 ml/min for 2 min at 25° C.

In the presence of antibody CLB-CAg A, a rise in the response wasactually observed (118 resonance units). Thus, the data clearlydemonstrate that Factor VIII A3-C1 is able to interact with LRP in adose-dependent manner.

TABLE VI Binding of Factor VIIIA3-C1 to immobilized LRP as detectedusing SPR analysis. Binding to LRP is expressed in resonance units andhas been corrected for nonspecific binding. Concentration A3-C1_(;)Binding (resonance units) 200 nM 47 400 nM 59 400 nM + 500 nM CLB-CAg A118

Example X Interaction Between Factor VIII A3-C1 and Cell-Surface-ExposedLow Density Lipoprotein Receptor-Related Protein

As described above, Factor VIII A3-C1 (i.e., residues 1649 to 2172) isable to interact with purified LRP. Furthermore, the interaction betweenFactor VIIIA3-C1 and LRPexpressed on the surface of living cells wasinvestigated. CHO-K1 cells stably expressing Factor VIII A3-C1 (asdescribed above) were, therefore, grown to confluence in 6 differentwells of a 24-well plate (Nunc A/S, Roskilde, Denmark). The wells werewashed five times using DMEM-F12 (Gibco-BRL, Breda, the Netherlands) and500 μl DMEM-F12 were added. In three of the wells, the LRP-antagonistRAP was added at a concentration of 1 μM at 2 and 4 hours after cellwashing. Samples were drawn up to 6 hours after cell washing and thenanalyzed for their Factor VIII A3-C1 concentrations. The concentrationsof Factor VIII A3-C1 were determined substantially using a methoddescribed in the art. (Lenting P. J. et al., J. Biol. Chem., Vol. 269,1994, pp. 7150-7155), except that the monoclonal antibody CLB-CAg 12 wasused instead of CLB-CAg 117. As illustrated in FIG. 8, the concentrationof Factor VIII A3-C1 increased with time in the absence of RAP. However,in the presence of RAP, the extent of Factor VIII A3-C1 rises ascompared to the absence of RAP. Thus, the inhibition of RAP isassociated with an accumulation of Factor VIII A3-C1 in the medium. Thisdemonstrates that an LRP-dependent mechanism is involved in the cellularuptake of Factor VI11 A3-C1.

FIG. 8 shows the effect of the LRP antagonist RAP on the concentrationof Factor VIII A3-C1 in a medium of Factor-VIII-A3-C1-expressing cells.In the absence of RAP (open symbols), the rise in Factor VIII A3-C1levels is lower than in the presence of RAP (closed symbols). The datarepresent the mean values±standard deviation of the three assays.

Example XI Mutations in the Factor VIII C2 Domain Affect Binding to LRP

As described in Example VII, the Factor VIII C2 domain comprises abinding site for LRP. Therefore, the effects of mutations in this domainon the binding of Factor VIII and the interaction withcell-surface-exposed LRP were studied. In order to express Factor VIIIvariants comprising such mutations, two constructs were prepared. BothFactor VIII expression plasmids were derivatives of the plasmidpF8-SQ#428 (F. Scheiflinger, unpublished results); a plasmid containingthe cDNA of a B-domain-deleted FVIII variant inserted in thecommercially available vector pSI (Promega). In this construct, all butfourteen amino acids of the B domain of FVIII were removed <SQ mutant,cf. Lind et al., Eur. J. Biochem., Vol. 232, 1995, p. 19-27).

Vector pF8-SQ#428 was modified by cutting with EcoRV/AgeI and ligatingthe annealed oligonucleotides P-A/Em(1) 5′-CCGGAGATTA TTACGAGGACAGTTATGAAG AC-3′ (SEQ ID NO: 6) and P-A/Em(2) 5′-GTCTTCATAA CTGTCCTCGTAATAATCT-3′ (SEQ ID NO: 7). This procedure resulted in vectorpF8-SQ-dA/E#501. Within this vector, the expression of FVIII-cDNA isdriven by the SV40 promoter and enhancer. Downstream, at the 3′ end ofthe FVIII gene, the polyadenylation site of SV40 is used to terminatethe transcription. A chimeric intron composed of the 5′-donor site fromthe first intron of the human β-globin gene and the branch and3′-acceptor site from the intron located between the leader and the bodyof a variable region of a heavy chain of an immunoglobulin gene wasintroduced to increase the level of gene expression (cf. pSI, productinformation, Promega). In order to further improve expression levels, asequence context found to be optimal for the initiation of eukaryoticprotein translation (“Kozak sequence” 5′-GCCACCATG-3′) was insertedimmediately upstream of the Factor VIII start codon (Kozak, J. Biol.Chem., Vol. 266, 1991, pp. 19867-19870). The vector pF8-SQ-dA/E#501 wasthen used to construct vectors pC2-m7#516 and pC2-m9#518.

The construction of pC2-m7#516 was effected by the insertion of annealedoligonucleotides Mu(1) 5′-CGAATTCACC CCCAGATTTG GGAACACCAG ATTGCCCTGAGGCTGGAGAT TCTGGGCTGC GAGGCACAGC AGCAGTACTG AGC-3¹ (SEQ ID NO: 8) andP-Mu(2) 5′-GGCCGCTCAG TACTGCTGCT GTGCCTCGCA GCCCAGAATC TCCAGCCTCAGGGCAATCTG GTGTTCCCAA ATCTGGGGGT GAATT-3′ {SEQ ID NO: 9) intoAsuII/NotI-cut vector pF8-SQ-dA/E#501.

The resulting vector encodes a Factor VIII variant comprising thefollowing substitutions: Ser²³¹² to Ile²³¹², Val²³¹⁴ to Glu²³¹⁴, Met²³²¹to Leu²³²¹, Val²³²³ to He²³²³, Asp²³³⁰ to Gln²³³⁰ and Leu²³³¹ toGln²³³¹.

The construction of pC2-m9#518 was effected by the insertion of annealedoligonucleotides P-CC(1) 5′-CTAGAACCAC CGTTAGTGGC TCGCTACGTG CGACTGCACCCCCAGAGTTG GGCTCACCAT-3′, P-CC(2) (SEQ ID NO: 10); 5′-ATTGCCCTGAGGCTGGAGGT TCTGGGCTGC GATACTCAGC AGCCAGCTTG AGC-3′, P-CC(3)(SEQIDNO:11).

5′-GGCCGCTCAA GCTGGCTGCT GAGTATCGCA GC-3′, P-CC(4) (SEQ ID NO: 12)5′-CCAGAACCTC CAGCCTCAGG GCAATATGGT GAGCCCAACT CTGGGGGTGC-3′ and P-CC(5)(SEQ ID NO: 13).

5′-AGTCGCACGT AGCGAGCCAC TAACGGTGGT T-3′ (SEQ ID NO: 14) intoXbaI/NotI-cut vector pF8-SQ-dA/E#501.

The construct pC2-m9#518 encodes a Factor VIII variant comprising thefollowing amino acid substitutions: Asp²²⁹⁸ to Glu²²⁹⁸, Leu²³⁰² toVal²³⁰², Thr²³⁰³ to Ala²³⁰³ and Leu²³⁰⁶ to Val²³⁰⁶, Ile²³⁰⁸ to Leu²³⁰⁸,Val²³¹⁴ to Ala²³¹⁴, Gln²³¹⁶ to His²³¹⁶, Mel²³²¹ to Leu²³²¹, Glu²³²⁷ toAsp²³²⁷, Ale²³²⁸ to Thr²³²⁸, Asp²³⁰⁰ to Gln²³³⁰, Leu²³³¹ to Pro²³³¹ andTyr²³³² to Ala²³³².

Both vectors pC2-m9#518 and pC2-m7#516 respectively encoding FactorVIII#518 and Factor VIII#516 were transfected into mouse fibroblast C127cells using calcium phosphate precipitation (J. Sambrook et al.,Molecular Cloning; A Laboratory Manual, Cold Spring Laboratory Press,Cold Spring Harbor, U.S.A., 1989, p. 1637). The vectors werecotransfected (20:1 ratio) using the plasmid pDH#310, thus allowing aselection of the transfectants with hygromycin B (200 μg/ml).

C127 cells stably expressing normal Factor VIM (cf. Example IV), FactorVIII#518 and Factor VIII#516 were grown to 50% confluence in 4 wells ofa 24-well plate (Nunc A/S, Roskilde, Denmark). The wells were washedfive times using IMEM (Boehringer Ingelheim/BioWhitaker, Verviers,Belgium) and 1 ml IMEM was added. In two of the wells for each FactorVIII variant, the LRP-antagonist RAP was added immediately to aconcentration of 20 mg/ml and 2 hours after cell washing. Samples weredrawn two and three hours after cell washing and then analyzed forFactor VIII cofactor activity using an established method (Mertens K. etal., Brit. J. Haematol., Vol. 85, 1993, 133-142). In Table VII, theFactor VIII expression levels in the presence and absence of RAP atdifferent points of time are indicated. For normal Factor VIII as wellas Factor VIII variants #516 and #518, an increase in the Factor VIIIactivity in the medium is observed in the presence or absence of RAP.For normal Factor VIII the expression level is, however, elevated in thepresence of RAP, whereas for Factor VIII variants #516 and #518 thiseffect is strongly reduced, if present at all. This demonstrates thatthe LRP-mediated cellular uptake of Factor VIM variants #516 and #518proceeds less efficiently than with normal Factor VIII. Thus, amino acidsubstitutions in the Factor VIII C2 domain as indicated for Factor VIIIvariants #516 and #518 render Factor VIII less sensitive to LRP-mediatedcellular uptake.

TABLE VII Expression levels of normal Factor VIII and Factor VIIIvariants #516 and #518 in the presence and absence of RAP. The datarepresent the ± mean values of two independent assays. Expression (U/L)Ratio Factor VIII Time (h) −RAP +RAP +RAP/−RAP Normal 2 1.3 ± 0.1 4.2 ±0.4 3.2 3 2.2 ± 0.2 5.0 ± 0.3 2.3 #516 2 0.3 ± 0.2 0.3 ± 0.2 1.0 3 0.6 ±0.1 0.8 ± 0.3 1.3 #518 2 1.1 ± 0.1 1.3 ± 0.4 1.2 3 3.3 ± 0.2 3.5 ± 0.31.1

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

This application claims priority to AT 1872/98 filed Nov. 10, 1998, theentirety of which is hereby incorporated by reference.

What is claimed is:
 1. A pharmaceutical preparation comprising: (A) amodified human Factor VIII polypeptide that has Factor VIII activity,wherein the modified human Factor VIII polypeptide differs from thehuman Factor VIII by at least one modification in at least one exositeof the A3 domain of said Factor VIII polypeptide, wherein said exositeis a hydrophilic moiety of the A3 domain of said modified human FactorVIII, and wherein the exosite is selected from the group of exosites,based upon the amino acid sequence of human Factor VIII polypeptide,consisting of the regions in said Factor VIII polypeptide: (i) 1743(Phe) to 1749 (Arg), (ii) 1784 (Ser) to 1831 (Asp), (iii) 1886 (Ser) to1919 (His), (iv) 1942 (Trp) to 1947 (Met), and (v) 1959 (Ser) to 1974(Ala), wherein the modification is an amino acid substitution, deletionor insertion, and wherein the modification reduces the binding affinityto low-density lipoprotein receptor-related protein (LRP); and (B) anadded lipoprotein receptor-related protein antagonist.
 2. Thepreparation according to claim 1, wherein the lipoproteinreceptor-related protein antagonists are selected from the groupconsisting of receptor-associated protein (RAP) and a fragment oflipoprotein receptor-related protein from clusters I, II, III or IV,wherein the fragment binds to a Factor VIII-LRP binding region.
 3. Thepreparation according to claim 1, wherein the modified human Factor VIIIpolypeptide further comprises at least one modification in at least oneexosite of the C2 domain, wherein exosite is a hydrophilic moiety of theC2 domain, and wherein the exosite is selected from the group ofexosites, based upon the amino acid sequence of human Factor VIIIpolypeptide, consisting of amino acid regions (i) 2209 (Arg) to 2234(Phe) and (ii) 2269 (His) to 2281 (Lys), wherein the modification is anamino acid substitution, deletion or insertion.
 4. The preparation ofclaim 1, wherein the modified human Factor VIII polypeptide is producedby recombinant techniques.
 5. A pharmaceutical preparation comprising:(A) a modified human Factor VIII polypeptide that has Factor VIIIactivity, wherein the modified human Factor VIII polypeptide differsfrom the human Factor VIII by at least one modification in at least oneexosite of the C1 domain, wherein said exosite is a hydrophilic moietyof the C1 domain, and wherein the exosite is selected from the group ofexosites, based upon the amino acid sequence of human Factor VIIIpolypeptide, consisting of the regions in said Factor VIII polypeptide:(i) 2037 (Ile) to 2062 (Trp), (ii) 2108 (Asp) to 2118 (Asn), and (iii)2154 (Thr) to 2158 (Ile), wherein the modification is an amino acidsubstitution, deletion or insertion, and wherein the modificationreduces the binding affinity to low-density lipoprotein receptor-relatedprotein (LRP); and (B) an added lipoprotein receptor-related proteinantagonist.
 6. The preparation according to claim 5, wherein thelipoprotein receptor-related protein antagonists are selected from thegroup consisting of receptor-associated protein (RAP) and a fragment oflipoprotein receptor-related protein from clusters I, II, III or IV,wherein the fragment binds to a Factor VIII-LRP binding region.
 7. Thepreparation according to claim 5, wherein the modified human Factor VIIIpolypeptide further comprises at least one modification in at least oneexosite of the C2 domain, wherein exosite is a hydrophilic moiety of theC2 domain, and wherein the exosite is selected from the group ofexosites, based upon the amino acid sequence of human Factor VIIIpolypeptide, consisting of amino acid regions (i) 2209 (Arg) to 2234(Phe) and (ii) 2269 (His) to 2281 (Lys), wherein the modification is anamino acid substitution, deletion or insertion.
 8. The preparation ofclaim 5, wherein the modified human Factor VIII polypeptide is producedby recombinant techniques.